Food or pet food composition containing plant extract for bone health

ABSTRACT

The present invention relates to a composition for maintenance of bone health or prevention, alleviation and/or treatment of bone disorders. It also relates to the use of the composition in the manufacture of a nutritional product, a supplement or a medicament; and a method of promoting bone growth or for the maintenance of bone health which comprises administering an effective amount of the composition.

This invention relates to a human or pet composition for maintenance ofbone health or prevention, alleviation and/or treatment of bonedisorders. It also relates to the use of the composition in themanufacture of a nutritional product, a supplement or a medicament; anda method of promoting bone growth or for the maintenance of bone healthwhich comprises administering an effective amount of the composition.

BACKGROUND OF THE INVENTION

Healthy bones require effective bone remodeling involving an equilibriumbetween bone formation and resorption. Most bone diseases are due toincreased bone resorption, rendering its inhibition a primarytherapeutic objective, therefore most pharmaceutical agents, developedto date, are anti-resorptive. For example, estrogens block production ofcytokines that promote osteoclast generation and differentiation. SERMs(selective estrogen response modulator), are being developed whichprovide benefits for bone health while reducing the risk of adversehormonal effects on breast or endometrial tissues. It is assumed thatthey work by a similar mechanism to estrogen in bone. Bisphosphonates(such as alendronate, risedronate etc) concentrate in bone and are, todate, the most effective inhibitors of bone resorption. They inhibit acritical enzyme pathway, required for osteoclast activity and survival.Calcitonin is a polypeptide hormone that inhibits bone resorption byblocking osteoclast activity. New targets include blocking of the TNFreceptor/ligand family members and their signalling pathways,particularly of RANK/RANKL, inhibition of bone-specificmetalloproteinases such as cathepsin K or inhibition of specifickinases.

Development of therapeutic agents stimulating bone formation has laggedbehind that of resorption. Some chemical or pharmaceutical agents areknown for promoting bone growth in human. For example, WO 9619246describes a method for promoting bone growth in a human patient by(normally intermittent not continuous) administration of parathyroidhormone, PTH-related protein or an agonist for at least one month. In WO9619501, a pancreatic-derived factor inhibits the resorption of bone andstimulates bone cells to proliferate and increases the formation ofbone.

Although these chemicals and pharmaceutical compounds have been provedfor the treatment of bone disorders, it would be of interest to providea safe, efficient nutritional way to promote bone growth and prevent oralleviate the symptoms of bone/joint disorders in mammals.

SUMMARY OF THE INVENTION

Accordingly, in a first aspect, the present invention provides acomposition intended for the prevention, alleviation and/or treatment ofbone disorders or maintenance of bone health in mammals, which comprisesas an active ingredient an effective amount at least one plant or plantextract selected from the group consisting of Taraxacum and Amelanchier.

Remarkably, it has now been found that some plants or plant extractshave a particular positive effect on bone formation and repair, onmaintenance of bone health or prevention, alleviation and/or treatmentof bone disorders.

The composition according to the invention can be used for themanufacture of a nutritional product, a supplement, a treat or amedicament intended for humans or pets.

Administering to a mammal a composition according to the presentinvention, results in an improved bone regeneration during fracturehealing. It helps to inhibit bone resorption. It also helps to increasebone formation and bone mineral density during growth and optimize peakbone mass. Also, this composition helps to decrease bone loss, inparticular bone loss associated with age in mammals. It reduces risk ofosteoporosis. Furthermore it helps to build cartilage in mammals, toprevent osteoarthritis in pets or humans, which results in a betteractivity or mobility of the individual.

In another aspect, the invention relates to the use of a plant or plantextract as described above, for the preparation of a compositionintended for the prevention, alleviation and/or treatment of bonedisorders or maintenance of bone health in mammals.

It also relates to the use of a plant or plant extract as describedabove, for the preparation of a composition intended for inhibiting boneresorption.

It further relates to the use of a plant or plant extract as describedabove, for the preparation of a composition intended for improvingactivity and/or mobility of pets or humans.

In addition, the invention provides a method for the treatment,alleviation and/or prophylaxis of bone disorder or maintenance of bonehealth in mammals which comprises administering an effective amount of acomposition as described above.

The invention further provides a method of increasing bone formation,bone mineral density during growth and optimize peak bone mass, treatingor preventing osteoporosis, stimulating bone regenaration duringfracture healing which comprises administering an effective amount of acomposition as described above.

It further relates to a method for the treatment, alleviation and/orprophylaxis of osteoarthritis in pets or in humans, comprising the stepof feeding an individual, a composition as described above.

It further provides a method decreasing bone loss, in particular boneloss associated with age in humans and pets, comprising the step offeeding an individual, a composition as described above.

Additional features and advantages of the present invention aredescribed in, and will be apparent from, the following DetailedDescription of the Invention and the figures.

DETAILED DESCRIPTION OF THE INVENTION

With respect to the first object of the present invention, the plant orplant extract is selected from the group consisting of Taraxacum andAmelanchier.

In a preferred embodiment, the plant or plant extract is Taraxacumofficinale (common dandelion), Taraxacum kok-saghyz or Amelanchierovalis, Amelanchier alnifolia, Amelanchier laevis, Amelanchier arborea,Amelanchier asiatica, for example.

The plant according to the invention may be from any part of the plantsource, e.g. leaves, tubers, fruit or roots. In a most preferredembodiment, leaves or roots of Taraxacum species, or fruits ofAmelanchier species, or a mixture thereof are used. The plant or plantextract may be in the form of a dried, lyophilized extract of leaves,roots and/or fruits depending on the source of plant, or fresh plant, orenriched fraction obtained by inorganic or organic solvant extractionprocess known in the art.

The plant or plant extract according to the invention may be used in thepreparation of a food composition. The said composition may be in theform of a nutritionally balanced food or pet food, a dietary supplement,a treat or a pharmaceutical composition.

The plant or plant extract may be used alone or in association withother plants such as chicory, tea, cocoa, or with other bioactivemolecule such as antioxidants, fatty acids, prebiotic fibers,glucosamine, chondroitin sulphate, for example.

In one embodiment, a food composition for human consumption is prepared.This composition may be a nutritional complete formula, a dairy product,a chilled or shelf stable beverage, soup, a dietary supplement, a mealreplacement, and a nutritional bar or a confectionery.

Apart from the plant extract according to the invention, the nutritionalformula may comprise a source of protein. Dietary proteins arepreferably used as a source of protein. The dietary proteins may be anysuitable dietary protein; for example animal proteins (such as milkproteins, meat proteins and egg proteins); vegetable proteins (such assoy protein, wheat protein, rice protein, and pea protein); mixtures offree amino acids; or combinations thereof. Milk proteins such as casein,whey proteins and soy proteins are particularly preferred. Thecomposition may also contain a source of carbohydrates and a source offat.

If the nutritional formula includes a fat source, the fat sourcepreferably provides about 5% to about 55% of the energy of thenutritional formula; for example about 20% to about 50% of the energy.The lipids making up the fat source may be any suitable fat or fatmixtures. Vegetable fats are particularly suitable; for example soy oil,palm oil, coconut oil, safflower oil, sunflower oil, corn oil, canolaoil, lecithins, and the like. Animal fats such as milk fats may also beadded if desired.

A source of carbohydrate may be added to the nutritional formula. Itpreferably provides about 40% to about 80% of the energy of thenutritional composition. Any suitable carbohydrates may be used, forexample sucrose, lactose, glucose, fructose, corn syrup solids, andmaltodextrins, and mixtures thereof. Dietary fibre may also be added ifdesired. If used, it preferably comprises up to about 5% of the energyof the nutritional formula. The dietary fibre may be from any suitableorigin, including for example soy, pea, oat, pectin, guar gum, gumarabic, and fructooligosaccharides. Suitable vitamins and minerals maybe included in the nutritional formula in an amount to meet theappropriate guidelines.

One or more food grade emulsifiers may be incorporated into thenutritional formula if desired; for example diacetyl tartaric acidesters of mono- and di- glycerides, lecithin and mono- anddi-glycerides. Similarly suitable salts and stabilisers may be included.Vitamins and minerals may also be combined with the plant extract.

The nutritional formula is preferably enterally administrable; forexample in the form of a powder, tablet, capsule, a liquid concentrate,solid product or a ready-to-drink beverage. If it is desired to producea powdered nutritional formula, the homogenised mixture is transferredto a suitable drying apparatus such as a spray drier or freeze drier andconverted to powder.

In another embodiment, a nutritional composition comprises a milk basedcereal together with a prebiotic formulation. Preferably the milk basedcereal is an infant cereal which acts as a carrier for the prebioticformulation.

In another embodiment, a usual food product may be enriched with atleast one plant or plant extract according to the present invention. Forexample, a fermented milk, a yoghurt, a fresh cheese, a renneted milk,article of confectionery, for example a sweet or sweetened beverage, aconfectionery bar, breakfast cereal flakes or bars, drinks, milkpowders, soy-based products, non-milk fermented products or nutritionalsupplements for clinical nutrition.

The amount of the plant or plant extract in the composition may varyaccording to the plant source and its utilization. In a preferredembodiment, an efficient daily dose amount is of at least about 1 mg,and more preferably from 1 mg to 200 mg of the active molecule per day.

In one embodiment, a pharmaceutical composition containing at least anextract or phytochemical as described above, in an amount sufficient toachieve the desired effect in an individual can be prepared. Thiscomposition may be a tablet, a liquid, capsules, soft capsules, pastesor pastilles, gums, or drinkable solutions or emulsions a dried oralsupplement, a wet oral supplement. The pharmaceutical composition willfurther contain carriers and excipients that are suitable for deliveringthe respective active molecule of different nature to the target tissue.The kind of the carrier/excipient and the amount thereof will depend onthe nature of the substance and the mode of drug delivery and/oradministration contemplated. It will be appreciated that the skilledperson will, based on his own knowledge select the appropriatecomponents and galenic form.

The plant or plant extract according to the invention may be used in thepreparation of a pet food composition. The said composition may beadministered to the pet as a supplement to its normal diet or as acomponent of a nutritionally complete pet food, and more preferably inan hypocaloric pet food. It may also be a pharmaceutical composition.

The plant or plant extract may be used alone or in association withother plants such as chicory, tea, cocoa, or with other bioactivemolecule such as antioxidants, fatty acids, prebiotic fibers,glucosamine, chondroitin sulphate for example.

Preferably, the pet food composition contains about 0.01 to 0.5 g of dryplants per gram of dry pet food for a 15 kg dog; and 0.001 to 0.1 g ofdry plants per gram of wet pet food for a 15 kg dog.

The nutritionally complete pet food composition according to theinvention may be in powdered, dried form, a treat or a wet, chilled orshelf stable pet food product. It may be chilled or provided as a shelfstable product. These pet foods may be produced by ways known in theart.

The pet food may optionally also contain a prebiotic, a probioticmicroorganism or another active agent, for example a long chain fattyacid. The amount of prebiotic in the pet food is preferably less than10% by weight. For example, the prebiotic may comprise about 0.1% toabout 5% by weight of the pet food. For pet foods which use chicory asthe source of the prebiotic, the chicory may be included to compriseabout 0.5% to about 10% by weight of the feed mixture; more preferablyabout 1% to about 5% by weight.

If a probiotic micro-organism is used, the pet food preferably containsabout 10⁴ to about 10¹⁰ cells of the probiotic micro-organism per gramof the pet food; more preferably about 10⁶ to about 10⁸ cells of theprobiotic micro-organism per gram. The pet food may contain about 0.5%to about 20% by weight of the mixture of the probiotic micro-organism;preferably about 1% to about 6% by weight; for example about 3% to about6% by weight.

If necessary, the pet food is supplemented with minerals and vitamins sothat they are nutritionally complete. Further, various otheringredients, for example, sugar, salt, spices, seasonings, flavouringagents, and the like may also be incorporated into the pet food asdesired.

In another embodiment, dietary adjuncts may be prepared so as to improvepet food quality. As dietary adjuncts, they may be encapsulated or maybe provided in powder form and packaged in conjunction with orseparately from a main meal, be it wet or dry. By way of example, apowder containing extracts according to the invention, may be packed insachets in a powder form or in a gel or lipid or other suitable carrier.These separately packaged units may be provided together with a mainmeal or in multi-unit packs for use with a main meal or treat, accordingto user instructions.

The amount of pet food to be consumed by the pet to obtain a beneficialeffect will depend on the size of the pet, the type of pet, and age ofthe pet. However, an amount of the pet food to provide a daily amount ofabout 0.5 to 5 g of dry plants per kg of body weight, would usually beadequate for dogs and cats.

Administering to a human or animal, the food or pet food composition asdescribed above, results in an improved bone regeneration duringfracture healing. It helps to stimulate bone formation and bone mineraldensity during growth and optimize peak bone mass. In particular it mayprovide an optimal bone growth during childhood for humans or pets. Thisfood composition helps to prevent bone loss, in particular bone lossassociated with age in mammals or bone loss associated with long termhospitalization. It reduces risk of osteoporosis. Furthermore it helpsto build cartilage in mammals, prevent osteoarthritis in humans andpets, which results in a better activity or mobility of the individual.

The following examples are given by way of illustration only and in noway should be construed as limiting the subject matter of the presentapplication. All percentages are given by weight unless otherwiseindicated. The examples are preceded by a brief description of thefigure.

FIG. 1: comparision of measured inhibition values for the Calvaria Assay(A) and the Pit Assay (B) for the extract of fruits of Amelanchieralnifolia (10 μg/ml): P.E. 734.

EXAMPLES Example 1 Effect of Amelanchier and Taraxacum Species on BoneFormation

Extracts of the plant species Amelanchier and Taraxacum were tested fortheir potential to induce bone formation or inhibit bone resorption.

Materials and Methods

Preparation of extracts for screening assays:

The ground plant material is defatted with hexane then extracted with amixture of alcohol and water, with different percentages of water from10 to 90%, preferably with 50%. The alcohols can be methyl or ethylalcohols, giving the extract la.

On an aliquot of the residue of this first extract, an enzymatichydrolysis is carried out with a and P glucosidases. Enzymes can bereplaced by acidic conditions. The operation may be done under mildconditions (room temperature) or through reflux with different acidconcentrations. The aqueous hydrolysed phase is extracted with anon-miscible solvent, preferably ethylacetate to give the extract 2a.

The extract can be dried, freeze-dried or supplied as a liquid form.

In some cases, polyphenols can be discarded through apolyvinylpolypyrrolidone (PVPP) treatment, avoiding artefact with thescreening assays.

Following the extract preparation, each extract was weighed, redissolvedin dimethylsulphoxide (DMSO) to a final concentration of 20 mg/ml andstored in aliquots at −20° C. This was used as a stock solution and wassubsequently diluted in media for each assay. A range of doses wastested in the assay systems. Subfractions were prepared by fractionationon reverse phase silica gel cartridge with elution by solvents ofvarying polarity

Bone Formation Assay

BMP-2 luciferase assay—The activity of the extracts was determined using2T3 cells containing the BMP-2 promoter operatively linked to theluciferase gene. An increase in luciferase activity in cell lysatesreflects an increase in BMP-2 promoter activity. The extracts wereassayed at an initial dilution of 100 μg/ml with 12 dilutions down to0.2 μg/ml. BMP-2 promoter activity was measured by measuring theluciferase activity in cell extracts.

Extracts of Amelanchier ovalis and Taraxacum officinalis gavesignificant positive results in stimulation of BMP-2 expression(Table 1) English conc Latin name name Part μg/ml Active extract/n°Amelanchier service Fruit 10 MeOH/water/219 ovalis berry Taraxacumcommon Leaves 50 ethylacetate/750/2034 officinalis dandelion andsubfraction 2035

Extracts of Amelanchier ovalis (P.E. 219 (MeOH/water)), and of Taraxacumofficinale (P.E. 750 (ethylacetate)) were further tested in a humanperiosteal/preosteoblast cell line, hPOB-tert for their ability toinduce the endogenous expression of BMP-2.

The validation of this assay was performed with statins as a positivecontrol. At confluence, cells were incubated with 0.5 μg/ml Lovastatinor with the plant extracts (10-50 μg/ml) for 24 h-48 h. Total RNA wasextracted with TRIzol Reagent (Gibco). 10 μg RNA were reversetranscribed using the 1st Strand cDNA Synthesis Kit (Boehringer). BMP-2cDNA sequences were amplified for 35 cycles at an annealing temperatureof 55° C. using specific oligonucleotide primers(5′:TTGCGGCTGCTCAGCATGTT; 3′:CATCTTGCATCTGTTCTCGGAA). PCR products wereseparated by agarose gel electrophoresis and detected by ethidiumbromide staining. Quantification was performed using NIH Image Softwareand normalizing results with Actin as house- keeping gene.

Results:

For Amelanchier ovalis (ext conc 10 μg/ml): induction of BMP-2 by 1.5fold.

For Taraxacum officinale (ext conc 50 μg/ml) induction of BMP-2 by 2.0fold.

Example 2 Effect of Amelanchier and Taraxacum Species on Bone Resorption

The ability of the extracts prepared as in example 1, to inhibit IL-1(10⁻¹⁰ M) stimulated bone resorption was assessed in the neonatal boneresorption assay. Each extract was assessed for its capacity to inhibitbone resorption at 10 μg/ml

Extracts of fruits of Amelanchier alnifolia (10 μg/ml) (P.E. 734(ethylacetate)) were able to inhibit IL-1 induced bone resorption in themurine calvaria assay ( R. J. Murills, “In vitro Bone Resorption Assays”in Principles of Bone Biology (Academic Press) 1986, chap. 90). Thiseffect was confirmed in a second bone resorption test, namely the pitassay using rabbit bone mixed cell cultures on bovine bone slices(Tezuka K., et al., 1992, Biochem. Biophys. Res. Commun. 186(2):911-7and Lorget F., et al., 2000, Biochem. Biophys. Res. Commun.268(3):899-903) Resorption pits are visualized by staining for TRAP(tartrate resistant acid phosphatase) positive cells and counted.

A comparison of activity of the extracts at 10 μg/ml in the two assaysystems is shown in FIG. 1.

Example 3 Dry Pet Food

A feed mixture is made up of about 58% by weight of corn, about 5.5% byweight of corn gluten, about 22% by weight of chicken meal, 2.5% driedchicory, about 10% of extract of Taraxacum leaves, salts, vitamins andminerals making up the remainder.

The feed mixture is fed into a preconditioner and moistened. Themoistened feed is then fed into an extruder-cooker and gelatinised. Thegelatinised matrix leaving the extruder is forced through a die andextruded. The extrudate is cut into pieces suitable for feeding to dogs,dried at about 110° C. for about 20 minutes, and cooled to form pellets.

This dry dog food has a positive effect on bone and cartillage healthand increase their mobility.

It should be understood that various changes and modifications to thepresently preferred embodiments described herein will be apparent tothose skilled in the art. Such changes and modifications can be madewithout departing from the spirit and scope of the present invention andwithout diminishing its intended advantages. It is therefore intendedthat such changes and modifications be covered by the appended claims.

1. A food composition intended for the prevention, alleviation and/ortreatment of bone disorders or maintenance of bone health in humans andpets comprising as an active ingredient an effective amount of at leastone plant or plant extract selected from the group consisting ofTaraxacum and Amelanchier.
 2. A composition according to claim 1,wherein the plant or plant extract is selected from the group consistingof Taraxacum officinale (common dandelion), Taraxacum kok-saghyz,Amelanchier ovalis, Amelanchier alnifolia, Amelanchier laevis,Amelanchier arborea and Amelanchier asiatica
 3. A composition accordingto claim 1 wherein the plant or plant extract is selected from the groupconsisting of leaves or roots of Taraxacum species, of fruits ofAmelanchier species, and mixtures thereof.
 4. A composition according toclaim 1 wherein the composition includes a component selected from thegroup consisting of chicory, tea, cocoa, other bioactive molecule suchas antioxidants, fatty acids, prebiotic fibers, glucosamine andchondroitin sulphate.
 5. A composition according to claim 1, wherein thecomposition is in a form selected from the group consisting of anutritionally balanced food, pet food, a dietary supplement, a treat anda pharmaceutical composition.
 6. A composition according to claim 1which helps bone regenaration during fracture healing, helps to increasebone formation and bone mineral density during growth and optimize peakbone mass or to decrease bone loss, in particular bone loss associatedwith age in humans or pets.
 7. A composition according to claim 1 whichis designed to build cartilage in humans or pets.
 8. A compositionaccording to claim 1 which is designed to prevent osteoarthritis inhumans or pets.
 9. A method for the preparation of a food or pet foodcomposition intended for the prevention, alleviation and/or thetreatment of bone disorders or maintenance of bone health in humans orpets comprising the steps of using a plant or a plant extract selectedfrom the group consisting of Taraxacum and Amelanchier to produce thecomposition.
 10. A method for the preparation of a food or pet foodcomposition intended to help bone regeneration during fracture healing,increase bone formation and bone mineral density during growth andoptimize peak bone mass or to decrease bone loss, in particular boneloss associated with age in humans or pets comprising the steps of usinga plant extract selected from the group consisting of Taraxacum andAmelanchier to produce the composition.
 11. A method for the preparationof a food or pet food composition intended for preventing or alleviatingthe symptoms of osteoarthritis in humans or pets comprising the steps ofusing a plant or a plant extract selected from the group consisting ofTaraxacum and Amelanchier to prepare the composition.
 12. A method forthe preparation of a food or pet food composition intended to assist inbuilding cartilage in humans or pets comprising the steps of using aplant extract selected from the group consisting of Taraxacum andAmelanchier to prepare the composition.
 13. (canceled)
 14. A method forthe treatment, alleviation or prevention of bone disorders ormaintenance of bone health in humans or pets, comprising the steps ofadministering an effective amount of a composition comprising as anactive ingredient an effective amount of at least one plant or plantextract selected from the group consisting of Taraxacum and Amelanchierto an individual at risk of a bone disorder.
 15. A method of increasingbone formation, bone mineral density during growth and optimize peakbone mass in humans or pets, comprising the step of feeding anindividual in need of same, a composition comprising as an activeingredient an effective amount of at least one plant or plant extractselected from the group consisting of Taraxacum and Amelanchier.
 16. Amethod for the treatment, alleviation and/or prophylaxis ofosteoarthritis in humans or pets, comprising the step of feeding to anindividual having or at risk for osteoarthritis, a compositioncomprising as an active ingredient an effective amount of at least oneplant or plant extract selected from the group consisting of Taraxacumand Amelanchier.
 17. A method of treating or preventing osteoporosis,comprising the steps of administering to an individual having or at riskof osteoporosis an effective amount of a composition comprising as anactive ingredient an effective amount of at least one plant or plantextract selected from the group consisting of Taraxacum and Amelanchier.18. A method of stimulating bone regenaration during fracture healing,comprising the step of feeding an individual having a fracture, a foodcomposition comprising as an active ingredient an effective amount of atleast one plant or plant extract selected from the group consisting ofTaraxacum and Amelanchier.
 19. A method of decreasing bone loss, inparticular bone loss associated with age in humans or pets, comprisingthe step of feeding to an individual having bone loss, a foodcomposition comprising as an active ingredient an effective amount of atleast one plant or plant extract selected from the group consisting ofTaraxacum and Amelanchier.